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目的 :构建糖多孢红霉菌KR6酶域基因失活突变体 ,探讨SRR氨基酸相应 9核苷酸去除突变体与合成酮内酯类化合物 3 脱氧 3 羰基 红霉内酯B的关系。方法 :以糖多孢红霉菌基因组DNA为模板 ,用重叠PCR技术扩增出缺少SRR氨基酸相应 9核苷酸的KR6酶域DNA片段 ,并克隆到载体pWHM3上 ,构建了同源重组质粒pWHM3 SRR。将pWHM3 SRR质粒转化糖多孢红霉菌λC3菌株 ,筛选出质粒整合到染色体上红霉素合成基因位点的整合体λC3 A、B和C。λC3 A在无硫链丝菌肽 (Thio)的R3M斜面上生长两代后 ,制备的原生质体涂R3M平皿 ,挑选出不能在含Thio的R3M斜面上生长、发酵液也无抑制枯草芽孢杆菌活性的突变菌株λC3 SRR。结果 :部分基因组DNA序列分析表明 ,糖多孢红霉菌λC3 SRR染色体上SRR氨基酸相应 9核苷酸已经敲除 ,ZabspecFab质谱分析证实λC3 SRR菌株合成了 3 脱氧 3 羰基 红霉内酯B。结论 :糖多孢红霉菌KR6酶域SRR氨基酸相应 9核苷酸敲除的λC3 SRR突变菌株可以合成 3 脱氧 3 羰基 红霉内酯B ,SRR为KR6酶域上NADPH 2′ 磷酸结合位点。
OBJECTIVE: To construct an inactivated mutant of KR6 enzyme domain of Saccharopolyspora rubra to investigate the relationship between the corresponding 9-nucleotide deletion mutant of SRR amino acid and deoxy-3-carbonyl erythronolide B, a synthetic ketolide compound. Methods: The DNA fragment of KR6 enzyme domain lacking the corresponding 9 nucleotides of SRR amino acids was amplified by overlap PCR using genomic DNA of Saccharopolyspora rubra as template, and cloned into vector pWHM3 to construct the homologous recombination plasmid pWHM3 SRR . The plasmid pWHM3 SRR was transformed into λC3 strain of Saccharopolyspora erythropolis and the integrants λC3 A, B and C with plasmids integrated into erythromycin synthesis gene sites on the chromosome were screened out. After growing two generations of λC3 A on the R3M slant of Thio, the prepared protoplasts were plated on R3M plates, picked out, unable to grow on R3M slants containing Thio, and the broth also did not inhibit Bacillus subtilis activity The mutant strain λC3 SRR. Results: Partial genomic DNA sequence analysis showed that the corresponding 9 nucleotides of the SRR amino acid of λC3 SRR was knocked out. The ZabspecFab mass spectrometry confirmed the synthesis of 3-deoxycarbonyl erythronolide B in λC3 SRR strain. CONCLUSION: 3R deoxycarbonyl 3-erythronolide B can be synthesized from the λC3 SRR mutant strain corresponding to the 9-nucleotide knock-out of the SRR amino acid of S. erythraea KR6 and SRR is the NADPH 2 ’phosphate binding site on the KR6 enzyme domain.